EP3274465A1

EP3274465A1 - Biocatalytic production of l-fucose

Info

Publication number: EP3274465A1
Application number: EP16705208.3A
Authority: EP
Inventors: Vaidotas NAVICKAS; Michael Breuer; Michael Puhl; Melanie WEINGARTEN; Kai-Uwe Baldenius; Wolfgang Siegel
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 2015-03-26
Filing date: 2016-02-19
Publication date: 2018-01-31
Anticipated expiration: 2036-02-19
Also published as: KR102584658B1; RU2017137403A; CN107454915B; JP2018509169A; WO2016150629A1; CA2978131A1; AU2016235568A1; DK3274465T3; AU2016235568B2; US20180073047A1; CN107454915A; US10428361B2; KR20170130559A; JP6695896B2; EP3274465B1; RU2017137403A3

Abstract

The invention relates to biocatalytic methods, including purely enzymatic, mixed enzymatic-fermentative and purely fermentative methods, for the direct single-step conversion of L-fucitol into L-fucose, in order to easily obtain L-fucose at high amounts and levels of purity. The invention also relates to suitable recombinant microorganisms and fungi, and to the use of same in said method for the single-step conversion of L-fucose.

Description

Biocatalytic production of L-fucose

Technical Field The present invention relates to methods for the biocatalytic, i. enzymatic and / or fermentative, production of L-fucose from L-fucitol. Furthermore, the present invention discloses enzymes, in particular a galactose oxidase, which are suitable for the biocatalytic production of L-fucose from L-fucitol. Also disclosed are recombinant microorganisms and fungi which are expressible as transgene and capable of expressing the nucleic acid sequences required for the enzymes for producing L-fucose from L-fucitol, and the use of these microorganisms or fungi in the methods according to the present invention Invention.

Background of the invention

Fucose occurs in two enantiomeric forms: L-fucose (also called isodecitic) and D-fucose (CAS 3615-37-0). The L-shape is the most common in nature. L-fucose is an elemental molecule for use in research and development and a characteristic component of glycan structures. Scientific studies in the field of fucosylated substances have shown, among other things, that they have an outstanding importance in embryonic development and are involved in the immune response of the organism. L-fucose is found in human breast milk, among others. Human breast milk is given an important role in healthy child development. The substances occurring therein, in particular the oligosaccharides (human milk oligosaccharides (HMO)) are one of the main solid components of breast milk and have a core structure which has a lactose unit at the reducing end and is branched or chain-continued with N-Acetyllactosamineinheiten. The structural variability is extended, inter alia, by fucosyl modifications at the end positions. In terms of health and development promoting effect, the biological function of HMOs is the subject of numerous studies, but requires the technical purification of the compounds in sufficient quantities. Currently, the delivery of L-fucose is either by direct extraction from natural sources or by chemical modification of monosaccharides (for a summary, see PT Vanhooren et al., J. Chem., Technol., Biotechnol., 74, 479 (1999), and therein by reference Sources). The elaborate extraction from mother's milk is currently the most widely used method. Although biotechnological production methods have been described for HMOs (see Han et al., Biotechnol. Adv. 2012, 30, 1268-1278), the corresponding HMOs are often not available at a reasonable price.

L-fucose and L-fucose containing substances are the subject of ongoing research. L-fucose itself and fucosylated substrates are important starting materials in the chemical and pharmaceutical industries as well as useful in the manufacture of cosmetics and nutraceuticals. Therefore, there is a constant need for innovative production processes for the production of L-fucose on an industrial scale. Biotechnological methods have been proposed to recover L-fucose. For example, WO 2012/034996 A1 teaches a process for the production of L-fucose using an isolated microorganism of the family Enterobacteriaceae (deposited as DSM 22227), which has a special 16S rRNA according to SEQ ID NO: 1, for the fermentative production of L. fucose. However, the L-fucose is obtained only after time-consuming fermentation as a non-purified mixture, which is why elaborate further purification steps are required to obtain L-fucose. Inter alia, US Pat. No. 8,642,297 B2 postulates a generic fermentative process which is intended to lead to the production of L-fucose using recombinant mannitol-1-dehydrogenase from Apium graveolens. However, neither a concrete embodiment nor an example is disclosed. Furthermore, no alternative enzyme is disclosed which could be used for the reaction. WO 2005/087941 A1 discloses a combined fermentative-enzymatic process for providing L-fucose. First, by using a dehydrogenase of an acetobacterium, L-fuculose from L- Produces fucitol. This must then be synthesized in a further step to L-fucose.

Therefore, there is still a great need to be able to produce L-fucose in sufficient purity in an economically feasible process in as few steps as possible in sufficient purity.

Summary of the invention

The present invention is therefore based on the object to provide a method for the biotechnological production of L-fucose and suitable enzymes and recombinant microorganisms and fungi, wherein L-fucose recovered directly in a one-step reaction from L-fucitol on an industrial scale can be.

The following invention solves this problem by providing a method using L-fucitol and galactose oxidase and optionally other enzymes selected from a peroxidase and / or a catalase, whereby the conversion of L-fucitol to L-fucose in a Ein Step reaction can be carried out and this high yields can be achieved. Further disclosed are suitable enzymes for this purpose. Finally, a recombinant microorganism or fungus is disclosed which can produce the enzymes relevant to the disclosed pathway and its use. Aspects and embodiments of the present invention will become apparent from the following detailed description and examples, the drawings, and the appended claims.

Brief description of the drawings

Figure 1 (Figure 1) shows the reaction pathway of galactose via L-fucitol to L-fucose causing the step of oxidation of L-fucitol by a galactose oxidase.

Figure 2 (Figure 2) shows the effect of substrate concentration on the conversion of L-fucitol to L-fucose using a galactose oxidase over time.

Figure 3 (Figure 3) shows the effect of H ₂ O ₂ on the conversion of L-fucitol to L-fucose using a galactose oxidase over time.

Figure 4 (Figure 4) shows the effect of L-fucose on the conversion of L-fucitol to L-fucose using a galactose oxidase over time.

Detailed description Definitions

The terms protein, polypeptide and enzyme are used interchangeably because of the always enzymatic function of the polypeptides disclosed herein.

The terms biocatalytic or biocatalysis as used herein refers to a reaction and acceleration or control of chemical reactions in which enzymes serve as biological catalysts. Biocatalysis can be carried out either by adding isolated enzymes (enzymatically) or directly in living cells (herein: fermentative).

The term recombinant microorganism or fungus as used herein refers to a microorganism or fungus which is a recombinant, i. (Partially) artificial biomolecule of interest, wherein the biomolecule may be a nucleic acid or a polypeptide sequence. Furthermore, the selected microorganism or fungal strain may carry mutations in its genome or on extended plasmid DNA as compared to an unmodified or wild-type strain which do not affect the nucleic acid sequence of the biomolecule of interest.

The term transgene, as used herein, refers to a nucleic acid molecule of a species which is inserted either directly into the genome of another species or by means of a vector outside the genome, but replicably into the other species. was brought. The modified organism is referred to as recombinant (see above).

The term progeny as used herein, in the context of a recombinant microorganism or fungus according to the present disclosure, refers to the progeny of such an organism that emerge from the parent organism by natural reproductive propagation processes, including sexual and asexual. It is known to those skilled in the art that in the course of reproduction mutations can naturally be introduced into the genome of an organism, whereby the progeny is genomically different from the parent organism but still assigned to the same (sub-) species can. Also, such naturally-altered progeny are therefore encompassed by the term progeny according to the present disclosure.

The term vector as used herein refers to a delivery vehicle for delivery of a nucleic acid molecule into a cell, microorganism or fungus. Vectors may be, among others, plasmids, cosmids or modified viruses. Further, the term vector is also intended to include agents which are suitable for direct introduction of a polypeptide into a cell or microorganism.

The term "express in functional form" as used herein refers to the ability of a recombinant microorganism or fungus to select one or more trans (s) selected from the group consisting of a galactose oxidase, a catalase and a peroxidase , as defined above, to give a polypeptide which, under suitable reaction conditions, satisfies the enzymatic function of a galactose oxidase, a catalase or a peroxidase, as set forth above.

Detailed description of the invention

In one aspect, the present invention provides a process for producing L-fucose, comprising the steps of: (a) providing L-fucitol, (b) providing a galactose oxidase of the class of enzymes EC 1.1.3.9, and optionally one or more further constituent (s), (c) oxidation of L-fucitol using a galactose oxidase and optionally the further constituent (s) and incubating the resulting mixture under conditions which limit the biocatalytic oxidation of L-fucitol to L-fucitol. Allow fucose, (d) optionally: isolating the synthesized L-fucose. The galactose oxidase (EC 1.1 .3.9) is a copper-dependent oxidase. It is often found in nature as an extracellular, monomeric enzyme secreted by many filamentous fungi. Galactose oxidases catalyze the oxidation of primary alcohols to the corresponding aldehydes, while molecular oxygen is simultaneously reduced to H ₂ O ₂ (see Paukner, R. et al., Galactose oxidase from Fusarium oxysporum expression in E.coli and P. pastoris and biochemical characterization; PLoS ONE, 2014, 9, e1001 16/1). The activity of a galactose oxidase can be determined by methods known to those skilled in the art, wherein the release of hydrogen peroxide (H ₂ O ₂ ) from reduction of oxygen (O ₂ ) in the presence of a suitable substrate (esp. Galactose) is quantified. Alternatively, the reduction of the O ₂ concentration is a measure of the activity of a galactose oxidase.

In one embodiment of this aspect, the method comprises the substeps (b1) providing a recombinant microorganism or fungus for recombinant expression of a galactose oxidase of the class of enzymes EC 1.1.3.9, wherein the microorganism or fungus is a transgene which is responsible for a galactose oxidase from a fungus the order of the Hypocreales, preferably selected from the genus Fusarium, in particular the species Fusarium oxysporum, encodes, (b2) cultivating the recombinant microorganism under conditions which allow the synthesis of galactose oxidase, (b3) optionally: isolating the synthesized galactose oxidase. The preferred galactose oxidase from Fusarium oxysporum according to the present disclosure is a polypeptide comprising 681 amino acids (GenBank® Accession Number: AHA90705.1). The corresponding nucleic acid sequence (2046 nucleotides including stop codon) of the coding sequence can be accessed with GenBank® as accession number KF601698.1.

It is known to those skilled in the art that any enzyme of class 1.1.3.9, defined by comparable catalytic performance, can be used in the process of the present invention as long as the enzyme adopts the primary alcohol oxidation function outlined above catalysed the corresponding aldehydes with simultaneous reduction of molecular oxygen to H ₂ 0 ₂ . It is also known to those skilled in the art that the reaction conditions may vary depending on the enzyme used. Adjustments to the reaction and / or culture conditions may be readily made by those skilled in the art.

In one embodiment of this aspect, the one or more optional further constituents are selected from the group consisting of peroxidases and / or catalases. According to any aspect of the present invention, the biocatalytic recovery of L-fucose from L-fucitol is taught by the use of a galactose oxidase and optionally in addition a peroxidase and / or a catalase. These biocatalytic reactions according to all aspects of the present invention can be carried out either completely enzymatically, enzymatically and fermentatively, or exclusively by fermentation. Enzymatically in this context means the use of isolated and optionally purified and further modified enzymes. Fermentative in this context means the expression of one or more targeting enzymes in a recombinant host organism under suitable reaction conditions which allow the expression of the enzyme in functional form.

Due to the fact that the reaction of conversion of L-fucitol to L-fucose using a galactose oxidase is an oxygen-dependent reaction, it is contemplated in all aspects and embodiments of the present invention that the reactants will always be in sufficient contact with Oxygen come. This oxygenation may be effected by means well known to those skilled in the art, such as aeration by gassing or venting a culture or reaction mixture.

As already mentioned, it is preferred if the one or more optional further ingredient (s) is / are selected from the group consisting of peroxidases and / or catalases. Catalases catalyze the reaction of the sum equation: 2 H ₂ 0 ₂ -> 0 ₂ + 2 H ₂ 0, and thus set the oxidase with L-fucitol formed in the reaction of the galactose-product H ₂ 0 ₂ in order. Peroxidases, such as horseradish peroxidase, are enzymes that catalyze the reduction of peroxides. The sum equation of the catalyzed reaction is also: 2 H ₂ 0 ₂ -> 0 ₂ + 2 H ₂ 0. The substrate specificity of peroxidases can vary widely. Horseradish peroxidase is an exemplary member of this class of enzymes with a wide range of donors and acceptors. Any catalase or peroxidase is suitable for use in the present invention, which under suitable reaction conditions can catalyze the decomposition of H ₂ O ₂ into oxygen and water.

It has been found that the addition of a peroxidase and / or a catalase advantageously influences the conversion of L-fucitol to L-fucose, since the H ₂ O ₂ resulting inter alia from the activity of the galactose oxidase by the activity of these additional H ₂ 0 ₂ detoxifying enzymes removed and thus the yield of L-fucose can be increased. In one embodiment, either a catalase or a peroxidase is used in addition to the galactose oxidase. In a preferred Embodiment, both a catalase and a peroxidase is used in addition to the galactose oxidase. The advantageous results of the last-mentioned embodiment are illustrated in FIG. 3. In one embodiment, the peroxidase is selected from a peroxidase of the group consisting of the enzyme class EC 1.1 1 .1.7, in particular from horseradish peroxidase (Armoracia rusticana). The horseradish peroxidase can either be obtained commercially (SIGMA-ALDRICH, eg product number: P8250) or be produced recombinantly. The GenBank accession numbers are X57564.1 for the nucleic acid sequence mRNA, and CAA40796.1 for the polypeptide sequence for the Armoracia rusticana enzyme.

In a further embodiment according to the first aspect of the present invention, the catalase is selected from a catalase of the group consisting of the enzyme class EC 1.1 1.1.6, in particular from bovine catalase (Bos taurus).

In one embodiment, the catalase bovine liver catalase is of the enzyme class EC 1.1 1.1.6 (source about SIGMA ALDRICH, product number: C30) which is suitable for direct enzymatic conversion. For recombinant expression, the nucleic acid sequence of an exemplary bovine liver catalase is publicly available as NCBI reference sequence NM_001035386.2 (mRNA), the corresponding translated polypeptide sequence is publicly available as NCBI reference sequence NP_001030463.1.

Similarly, for all aspects of the fermentative production of enzymes in accordance with the present disclosure, galactose oxidase and / or peroxidase and / or catalyzer sequences will also have sequences of at least 80%, 81%, 82%, 83%, 84%, 85%, 86% %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the database sequences listed above, wherein the nucleic acid or polypeptide sequences may be codon-optimized, or may contain targeted point mutations, as long as the translated polypeptide still fulfills the function of the wild-type reference sequence. Such modifications for the adaptation of the nucleic acid and thus the polypeptide sequence of an enzyme are well known to the person skilled in the art and serve, inter alia, for example the adaptation of the sequence to the codon usage of the recombinant host organism selected for the expression, the optimization of the enzyme stability or function as well as the Increasing the yield or lowering the cytotoxicity of the enzyme product for the chosen host organism.

Furthermore, the nucleic acid or polypeptide sequences of the present disclosure may carry tagging sequences ("tags") which facilitate the purification or detection of the permit translated polypeptide sequences which are translated as a fusion molecule with the tag. Such tags and their sequences as well as methods for modifying a target sequence are well known to those skilled in the art and include sequences such as streptavidin tag, poly histidine tag, glutathione S-transferase tag, maltose binding protein tag, flash tag , Tags mediating the secretion of the recombinant polypeptide into the culture supernatant, and the like.

In one embodiment of the present invention, the concentration of galactose oxidase in the mixture at the beginning of the incubation in step (c) according to the first aspect is in the range of 0.005 to 6.25 g / L, preferably in the range of 0.005 to 3.2 g / L, more preferably in the range of 0.005 to 0.5 g / L and most preferably in the range of 0.005 to 0.05 g / L. It is known to those skilled in the art that the concentration of galactose oxidase used depends on the specific enzyme used and its specific enzyme activity. The enzyme activity can be given in two possible units: (1) in units (1 enzyme unit) = Ι μητιοΙ substrate min ^"1 , or as a unit of unit in Katal (kat) = 1 mol of substrate s ^' Approximately calculating the rate of an enzymatic reaction and thus also the enzyme activity by the Michaelis-Menten equation known, so that in a suitable assay, the enzyme activity for an enzyme of interest can be easily determined.

In another embodiment of the present invention, the concentration of peroxidase in the mixture at the beginning of the incubation in step (c) of the first aspect is in the range of 0 to 0.5 g / L, preferably in the range of 0 to 0.3 g In another embodiment of the present invention, the concentration of catalase in the mixture at the beginning of the incubation in step (c) of the first aspect is in the range of zero to 0.4 g / L, preferably in the range of 0 to 0.2 g / L, and more preferably in the range of 0 to 0.1 g / L. In yet another embodiment of the present invention, the concentration of Fucitol in the mixture at the beginning of the incubation in step (c) according to the first aspect in the range of 1 to 500 g / L, preferably in the range of 5 to 250 g / L, particularly preferably in the range of 20 to 125 g / L. In an embodiment according to any aspect of the present invention Invention is the recombinant microorganism or fungus selected from the group consisting of Escherichia coli spp., Preferably E. coli BL21, E. coli MG1655 or E. coli W31 10 and their derivatives, Bacillus spp., preferably Bacillus licheniformis, Bacillus subitilis or Bacillus amyloliquefaciens, and their derivatives, Saccharomycetes, preferably the order of Saccharomycetales, more preferably the family of Saccharomycetaceae, more preferably the genus of Komagataella, preferably Hansenula or Pichia spp. , more preferably P. pastoris and its derivatives, Kluyveromyces spp, preferably K. lactis, and their derivatives, Aspergillus spp., preferably A. oryzae, A. nidulans, or A. niger, and their derivatives, or Trichoderma spp., preferred T. reesei or T. harzianum, and their progeny, or a fungus of the Ascomycota Division, prefers Myceliopthora thermophila, or a derivative thereof.

According to another aspect of the present invention, there is provided a recombinant microorganism or fungus, wherein the microorganism or fungus contains a transgene encoding a galactose oxidase from a fungus of the genus Fusarium, and further a transgene selected from a peroxidase and / or a catalase as defined above, wherein the recombinant microorganism or fungus is selected from the group consisting of Escherichia coli spp., preferably E. coli BL21, E. coli MG1655 or E. coli W31 10 and their derivatives, Bacillus spp., Preferred Bacillus licheniformis, Bacillus subitilis or Bacillus amyloliquefaciens, and their derivatives, Saccharomycetes, preferably the order of Saccharomycetales, more preferably the family of Saccharomycetaceae, more preferably the genus of Komagataella, preferably Hansenula or Pichia spp., Particularly preferably P. pastoris and their Derivatives, Kluyveromyces spp, preferably K. lactis, and de descendants, Aspergillus spp., preferably A. oryzae, A. nidulans, or A. niger, and their progeny, or Trichoderma spp., preferably T. reesei or T. harzianum, and their progeny, or a fungus of the Ascomycota Division , preferably Myceliopthora thermophila, or a derivative thereof, and wherein the recombinant microorganism or fungus can express the transgene (s) in functional form. It is well known to those skilled in the art that the term "suitable (reaction) conditions" in the context of enzymes implies that the necessary substrates, buffering conditions, especially salt concentrations, optionally cofactors including, but not limited to, prosthetic groups, coenzymes and metal And provide pH and temperature conditions which are indispensable or conducive to the activity of the corresponding enzyme, for example, in the case of using a galactose oxidase - an oxygen-dependent enzyme - this includes aeration by gassing or venting a culture or reaction mixture, since all of the enzymes of the present invention as well as the reaction catalyzed by them are well characterized Therefore, the determination of suitable reaction conditions is within the skill of those in the art.

In one embodiment, besides the transgene encoding a galactose oxidase, another transgene encoding a catalase is contained in the recombinant microorganism or fungus.

In another embodiment, in addition to the transgene which codes for a galactose oxidase, another transgene which codes for a peroxidase is contained in the recombinant microorganism or fungus.

In yet another embodiment, besides the transgene encoding a galactose oxidase, both another transgene encoding a peroxidase and a transgene encoding a catalase are contained in the recombinant microorganism or fungus.

The provision of the particular transgene, its introduction into the recombinant microorganism or fungus, as well as the selection of suitable culture and reaction conditions which allow transcription and translation of the particular transgene, are well known to those skilled in the art.

In one embodiment, the recombinant microorganism or fungus may be used by adding L-fucitol to the culture medium for direct fermentative production of L-fucose from L-fucitol, i. the recombinant microorganism or fungus is used directly to carry out the method described above. This embodiment allows the direct production of L-fucose, optionally followed by further purification steps, in the fermentation batch.

In one embodiment, the galactose oxidase transgenes, as well as the peroxidase and / or catalase, can be tagged to allow secretion of the translated polypeptides into the culture supernatant. This allows either the easy isolation of the enzymes from the culture supernatant or the direct implementation of the conversion of L-fucitol to L-fucitose after addition of L-fucitol and the adaptation of the reaction conditions so that the secreted enzymes can fulfill their enzymatic function.

In another embodiment, the transgenes which code for a galactose oxidase, a peroxidase and / or a catalase are not provided in one but in different recombinant microorganisms or fungi and under suitable culture conditions and optionally purified. Then, with the enzymes thus obtained, in vitro or in vivo, after addition of L-fucitol, the procedure described herein can be carried out so that L-fucose can be obtained from L-fucitol by providing suitable reaction conditions.

In another aspect, the present invention provides the use of a recombinant microorganism or fungus, wherein the microorganism or fungus contains a transgene encoding a galactose oxidase from a fungus of the genus Fusarium, as defined above, for recombinant expression a galactose oxidase, in a process as above wherein the recombinant microorganism or fungus is selected from the group consisting of Escherichia coli spp., preferably E. coli BL21, E. coli MG1655 or E. co // ^'W31 10 and their derivatives, Bacillus spp., preferably Bacillus licheniformis, Bacillus subitilis or Bacillus amyloliquefaciens, and their derivatives, Saccharomycetes, preferably the order of Saccharomycetales, more preferably the family of Saccharomycetaceae, more preferably the genus of Komagataella, preferably Hansenula or Pichia spp. , more preferably P. pastoris and their derivatives, Kluyveromyces spp, preferably K lactis, and their derivatives, Aspergillus spp., preferably A oryzae, A. nidulans, or A. niger, and their derivatives, or Trichoderma spp., preferably T. reesei or T. harzianum, and their descendants, or a Ascomycota fungus is, preferably Myceliopthora thermophila, or a derivative thereof, and wherein the recombinant microorganism or fungus can express the transgene (s) in functional form.

In a further aspect, the present invention provides the use of a galactose oxidase and at least one further enzyme selected from a peroxidase and / or a catalase, wherein the galactose oxidase as well as the peroxidase and / or the catalase is preferably obtained by a method comprising the following steps:

(A) providing a recombinant microorganism or fungus for recombinant expression of a galactose oxidase and a peroxidase and / or a catalase, wherein the microorganism contains a transgene encoding a galactose oxidase from a fungus of the genus Fusarium, and a another transgene coding for a peroxidase and / or catalase, and wherein the recombinant microorganism or fungus is selected from the group consisting of Escherichia coli spp., preferably E. coli BL21, E. coli MG1655 or E. coli W31 10 and their derivatives, Bacillus spp., preferably Bacillus licheniformis, Bacillus subitilis or Bacillus amyloliquefaciens, and their derivatives, Saccharomycetes, preferably the order of Saccharomycetales, more preferably the family of Saccharomycetaceae, more preferably the genus of Komagataella, preferably Hansenula or Pichia spp ., more preferably P. pastoris and their derivatives, Kluyveromyces spp, preferably K. lactis, and their derivatives, Aspergillus spp., preferably A. oryzae, A. nidulans, or A. niger, and their progeny, or Trichoderma spp., preferably T Reesei or T. harzianum, and their progeny, or a fungus of the Ascomycota Division, prefers Myceliopthora thermophila, or a derivative thereof, or

(b1) providing a first recombinant microorganism or fungus for recombinant expression of a galactose oxidase, said microorganism or fungus containing a transgene encoding a galactose oxidase from a fungus of the genus Fusarium; and

(b2) providing a second recombinant microorganism or fungus for recombinant expression of a peroxidase, said microorganism or fungus containing a transgene encoding a peroxidase; and or

(b3) providing a third recombinant microorganism or fungus for recombinant expression of a catalase, said microorganism or fungus containing a transgene encoding a catalase;

(c) cultivating the recombinant microorganism under suitable conditions permitting the synthesis of galactose oxidase and peroxidase and / or catalase,

(d) optionally: isolating the synthesized galactose oxidase and the peroxidase and / or the catalase, for the biocatalytic conversion of L-fucitol to L-fucose, in a method according to the first aspect of the present invention.

For the peroxidase and / or catalase, therefore, the above applies accordingly.

This aspect of the present invention is especially useful to provide, prepare, and optionally isolate all desirable single enzymes for performing biocatalytic conversion of L-fucitol to L-fucose individually in one or more recombinant organisms, to facilitate the conversion of L-fucitol to L-fucose. Fucitol to L-fucose subsequently selectively enzymatically perform in vitro.

Examples

The invention will now be illustrated in more detail by way of non-limiting examples.

Example 1: Production of L-fucose by biocatalvic oxidation of L-fucitol with galactose oxidase in the presence of peroxidase and catalase

To a round-bottomed three-necked flask (50 mL) was added a solution of L-fucitol (6.0 mL aqueous solution containing 600 mg L-fucitol, CAS 13074-06-1, Santa Cruz Biotechnology), followed by addition of 1, 2 mL K ₂ HPO ₄ / KH ₂ PO ₄ (1000 mM, pH = 7.0) and 0.095 mL catalase (from bovine liver, SIGMA, 21.300 U / mg, 34 mg / mL), add 0.120 mL peroxidase (from horseradish , 173 U / mg, SIGMA) and 2.218 mL galactose oxidase (38.4 mg / mL, 2,708 U / mL). The resulting solution was sparged with O ₂ at room temperature until all L-fucitol had been converted to L-fucose. The reaction was monitored by HLPC. The final product was isolated and analyzed by 1 H and 13 C NMR. The results are summarized in Table 1. [Table 1]

Reaction time [h] conversion [%]

0

3.5 54.0

24 95.8

29 96.0

Example 2: Influence of peroxidase and catalase on the biocatalvic oxidation of L-fucitol

The following experiments were performed on a 1.0 mL scale: galactose oxidase (GO): To a 0.250 mL L-Fucitol solution (100 mg / mL) was added 0.635 mL water, followed by 0.050 mL K2HPO4 / KH2PO4 buffer, pH 7 0.055 mL galactose oxidase (2,705 U / mL in 100 mM K3PO4 buffer, pH 6.0); GO + catalase: To a 0.250 mL L-Fucitol solution (100 mg / mL) was added 0.631 mL water, followed by 0.050 mL K2HPO4 / KH2PO4 buffer, pH 7.0, 0.004 mL catalase in water (724,200 U / mL), 0.065 mL galactose oxidase (2,705 U / mL in 100 mM K3PO4 buffer, pH 6.0) added GO + peroxidase: to a 0.250 mL L-fucitol solution (100 mg / mL) were 0.630 mL of water followed by 0.050 mL K2HPO4 / KH2PO4 buffer, pH 7.0, 0.005 mL peroxidase in water (7.500 U / mL), 0.065 mL galactose oxidase (2.705 U / mL in 100 mM K3PO4 buffer, pH 6.0). , added; GO + peroxidase + catalase: To a 0.250 mL L-Fucitol solution (100 mg / mL) was added 0.626 mL water followed by 0.050 mL K2HPO4 / KH2PO4 buffer, pH 7.0, 0.005 mL peroxidase in water (7.500 U / mL) , Added 0.004 mL catalase in water (724,200 U / mL), 0.065 mL galactose oxidase (2,705 U / mL in 100 mM K3PO4 buffer, pH 6.0). All reactions were purged with oxygen and stirred for 60 min. The course of the reaction was monitored by HPLC. The results of the different reactions are summarized in FIG.

Example 3: Influence of the L-fucitol concentration on the biocatalytic oxidation of L-fucitol The following experiments were carried out on a 1.0 mL scale:

25 g / L L-fucitol: To a 0.100 mL L-Fucitol solution (100 mg / mL) was added 0.231 mL water, followed by 0.040 mL K2HPO4 / KH2PO4 buffer, pH 7.0, 0.005 mL peroxidase in water (7500 U / mL). mL), 0.004 ml catalase in water (724,200 U / mL), 0.065 mL galactose oxidase (2,705 U / mL in 100 mM in K3PO4 buffer, pH 6.0). 50 g / L L-fucitol: 0.020 g L-fucitol was added to 0.281 mL water, followed by 0.040 mL K2HPO4 / KH2PO4 buffer, pH 7.0, 0.004 mL peroxidase in water (7500 U / mL), 0.0032 mL catalase in water (724,200 U / mL), 0.052 mL galactose oxidase (2,705 U / mL in 100 mM in K3PO4 buffer, pH 6.0). 100 g / L L-fucitol: 0.040 g L-fucitol was added to 0.202 mL water, followed by 0.040 mL K2HPO4 / KH2PO4 buffer solution pH 7.0, 0.0080 mL peroxidase in water (7.500 U / mL), 0, Added 0064 ml of catalase in water (724,200 U / mL), 0.10 mL of galactose oxidase (2,705 U / mL in 100 mM in K3PO4 buffer, pH 6.0). 200g / L L-fucitol: 0.080g L-fucitol was added to 0.044mL water, followed by 0.040mL K2HPO4 / KH2PO4 buffer, pH 7.0, 0.0160mL peroxidase in water (7500U / mL), 0.0127mL Catalase in water (724,200 U / mL), 0.207 mL galactose oxidase (2,705 U / mL in 100 mM K3PO4 buffer, pH 6.0). All reactions were purged with oxygen and stirred for 60 min. The course of the reaction was monitored by HPLC. The results are summarized in FIG.

FIG. 4 shows a further control experiment in which the influence of L-fucose on the conversion of L-fucitol was additionally investigated. Example 4: Fermentative production and recovery of galactose oxidase

Pichia pastoris was probed with a suitable vector into which the wild-type galactose oxidase gene of Fusarium spp. was cloned, transformed. As a vector, any available Pichia compatible vector proved useful. In addition, different tags were used, either to aid in the purification of the resulting enzyme and / or to cause its secretion into the culture supernatant. As the promoter, a methanol-inducible promoter was chosen. This was shown on the fermenter scale for Pichia as a host organism suitable for inducing the expression of large amounts of galactose oxidase. The resulting galactose oxidase was optionally isolated or recovered from the culture supernatant and optionally purified. The recovered galactose oxidase had an activity of about 3,000 U / g. In the case of using a filamentous fungus as a recombinant fungus, the secretion of the galactose oxidase was also effected directly in the culture supernatant.

Further examples were made with other expression strains, including E. coli, and various well-known fungal strains and the vectors, controls, media and culture conditions used were adjusted accordingly. These modifications are well known to those skilled in the art. All of these expressions produced galactose oxidase in sufficient quantity, purity and with sufficient activity to catalyze the biocatalytic production of L-fucose. Particularly preferred strains have been found to be yeast and fungal strains.

Claims

claims

A process for producing L-fucose comprising the steps of:

(a) providing L-fucitol,

(b) providing a galactose oxidase of the enzyme class EC 1.1.3.9 and optionally one or more further constituents,

(c) oxidizing L-fucitol using the galactose oxidase and optionally the further constituent (s) and incubating the resulting mixture under conditions allowing the biocatalytic oxidation of L-fucitol to L-fucose,

(d) optionally: isolating the synthesized L-fucose.

2. The method of claim 1, wherein the one or more optional further ingredient (s) is / are selected from the group consisting of a peroxidase and / or a catalase.

3. The method according to claim 1 or 2, wherein step (b) comprises the following substeps or consists of:

(b1) providing a recombinant microorganism or fungus for recombinant expression of a galactose oxidase of the class of enzymes EC 1.1.3.9, wherein the microorganism or fungus is a transgene selected for a galactose oxidase from a fungus of the order Hypocreales, preferably from the genus Fusarium, in particular the species Fusarium oxysporum, encoded contains,

(b2) cultivating the recombinant microorganism under conditions such that

Allow synthesis of a galactose oxidase,

(b3) optionally: isolating the synthesized galactose oxidase.

4. The method of claim 3, wherein the recombinant microorganism or fungus is selected from the group consisting of Escherichia coli spp., Preferably E. coli BL21, E. coli MG1655 or E. coli W31 10 and their derivatives, Bacillus spp., Preferred Bacillus licheniformis, Bacillus subitilis or Bacillus amyloliquefaciens, and their derivatives, Saccharomycetes, preferably the order of Saccharomycetales, more preferably the family of Saccharomycetaceae, more preferably the genus of Komagataella, preferably Hansenula or Pichia spp., Particularly preferably P. pastoris and their Derivatives, Kluyveromyces spp, preferably K. lactis, and their derivatives, Aspergillus spp., Preferably A. oryzae, A. nidulans, or A. niger, and their derivatives, or Trichoderma spp., Preferably T. reesei or T. harzianum, and their abatement or Ascomycota fungus is, preferably Myceliopthora thermophila, or a derivative thereof.

5. The method according to any one of claims 2 to 4, wherein the peroxidase is selected from a peroxidase of the group consisting of the enzyme class EC 1.1 1.1.7, in particular from the horseradish peroxidase (Armoracia rusticana).

6. The method according to any one of claims 2 to 5, wherein the catalase is selected from a catalase of the group consisting of the enzyme class EC 1.1 1 .1.6, in particular from bovine catalase (ßos taurus).

7. The method according to any one of the preceding claims, wherein the concentration of L-fucitol in the mixture at the beginning of the incubation in step (c) in the range of 1 to 500 g / L, preferably in the range of 5 to 250 g / L, more preferably in the range of 20 to 125 g / L.

8. The method according to any one of the preceding claims, wherein the concentration of galactose oxidase in the mixture at the beginning of the incubation in step (c) in the range of 0.005 to 6.25 g / L, preferably in the range of 0.005 to 3.2 g / L, more preferably in the range of 0.005 to 0.5 g / L and most preferably in the range of 0.005 to 0.05 g / L.

9. The method according to any one of claims 2 to 8, wherein the concentration of peroxidase in the mixture at the beginning of the incubation in step (c) in the range of 0 to 0.5 g / L, preferably in the range of 0 to 0.3 g / L, and more preferably in the range of 0 to 0.15 g / L.

10. The method according to any one of claims 2 to 9, wherein the concentration of catalase in the mixture at the beginning of the incubation in step (c) in the range of 0 to 0.4 g / L, preferably in the range of 0 to 0.2 g / L, and more preferably in the range of 0 to 0.1 g / L.

1 1. A recombinant microorganism or fungus, wherein the microorganism or fungus contains a transgene encoding a galactose oxidase from a fungus of the genus Fusarium as defined in claim 3, and further a transgene selected from a peroxidase as in claim 5 and / or a catalase as defined in claim 6, wherein the recombinant microorganism or fungus is selected from the group consisting of Escherichia coli spp., preferably E. coli BL21, E. coli MG1655 or E. co /; ^' W31 10 and their offspring, Bacillus spp., Preferably Bacillus licheniformis, Bacillus subitilis or Bacillus amyloliquefaciens, and their derivatives, Saccharomycetes, preferably the order of Saccharomycetales, more preferably the family of Saccharomycetaceae, more preferably the genus of Komagataella, preferably Hansenula or Pichia spp., Particularly preferably P. pastoris and their progeny, Kluyveromyces spp, prefers K. lactis, and their progeny, Aspergillus spp., Preferably A. oryzae, A. nidulans, or A. niger, and their progeny, or Trichoderma spp., Preferably T. reesei or T. harzianum, and their progeny, or a fungus of the Ascomycota Division, preferably Myceliopthora thermophila, or a derivative thereof, and wherein the recombinant microorganism or fungus can express the transgene (s) in functional form.

12. Use of a recombinant microorganism or fungus according to claim 1 1 in a method according to any one of claims 1 to 10.

Use of a recombinant microorganism or fungus, wherein the microorganism or fungus contains a transgene encoding a galactose oxidase from a fungus of the genus Fusarium as defined in claim 3 for recombinant expression of a galactose oxidase in a process according to any one of claims 1 to 10, wherein the recombinant microorganism or fungus is selected from the group consisting of Escherichia coli spp., preferably E. coli BL21, E. coli MG1655 or E. co /; ^' W31 10 and their derivatives, Bacillus spp., Preferably Bacillus licheniformis, Bacillus subitilis or Bacillus amyloliquefaciens, and their derivatives, Saccharomycetes, preferably the order of Saccharomycetales, more preferably the family of Saccharomycetaceae, more preferably the genus of Komagataella, preferably Hansenula or Pichia spp., More preferably P. pastoris and its derivatives, Kluyveromyces spp, preferably K. lactis, and their derivatives, Aspergillus spp., Preferably A. oryzae, A. nidulans, or A. niger, and their progeny, or Trichoderma spp., preferably T. reesei or T. harzianum, and their progeny, or a fungus of the Ascomycota Division, prefers Myceliopthora thermophila, or a derivative thereof, and wherein the recombinant microorganism or fungus is the transgene (s) in functional form can express.

14. Use of a galactose oxidase and at least one further enzyme selected from a peroxidase and / or a catalase, the galactose oxidase and the peroxidase and / or the catalase preferably being obtained by a process comprising the following steps: (a) providing a recombinant microorganism or fungus for recombinant expression of a galactose oxidase and a peroxidase and / or a catalase, the microorganism containing a transgene coding for a galactose oxidase from a fungus of the genus Fusarium, and another transgene encoding a peroxidase and / or catalase, and wherein the recombinant microorganism or fungus is selected from the group consisting of Escherichia coli spp., preferably E. coli BL21, E. coli MG1655 or E. coli W31 10 and their derivatives, Bacillus spp., Preferably Bacillus licheniformis, Bacillus subitilis or Bacillus amyloliquefaciens, and their derivatives, Saccharomycetes, preferably the order of Saccharomycetales, more preferably the family of Saccharomycetaceae, more preferably the genus of Komagataella, preferably Hansenula or Pichia spp., More preferably P. pastoris and its derivatives, Kluyveromyces spp, bevo K. lactis, and their derivatives, Aspergillus spp., preferably A. oryzae, A. nidulans, or A. niger, and their derivatives, or Trichoderma spp., preferably T. reesei or T. harzianum, and their derivatives, or a fungus of the Ascomycota Division, preferably Myceliopthora thermophila, or a derivative thereof, or

(b2) providing a second recombinant microorganism or fungus for recombinant expression of a peroxidase, said microorganism or fungus containing a transgene encoding a peroxidase as defined in claim 5; and or

(b3) providing a third recombinant microorganism or fungus for recombinant expression of a catalase, said microorganism or fungus containing a transgene encoding a catalase as defined in claim 6;

(d) optionally: isolating the synthesized galactose oxidase and the peroxidase and / or the catalase,

for the biocatalytic conversion of L-fucitol to L-fucose, in a method according to one of claims 1 to 10.